E coli polar lipids extract

C-terminally His-tagged proteins were produced in E. coli BL21(DE3) cells and purified using immobilized Ni²⁺ affinity chromatography, as previously described [56 ]. Reconstitution of purified protein into proteoliposomes was performed using E. coli polar lipids extract (Avanti Polar Lipids, Alabaster, AL, USA) at a calculated LPR of 10 or 50, as previously described [38 (link)]. As previously shown [57 (link)], an LPR of 10 corresponts to a protein density of ~ 1000 protein particles/μm².

Planar lipid membrane experiments were performed as described previously^{92 (link)}. The electrolyte solution contained 1 M KCl and 10 mM Tris-HCl (pH 7.4). In all, 5 µl of purified protein (80 ng/ml) was mixed with 1500 µl KCl and was added to the cis compartment with a positive electrode, whereas the trans compartment was grounded. Planar lipid membrane was formed across a 0.5-mm aperture by painting the E. coli polar lipids extract (3% wt/vol, Avanti Polar Lipids, Alabaster, USA) dissolved in n-decan and butanol (9:1). The membrane current was registered using Ag/AgCl electrodes with salt bridges connected to an LCA-200-10G amplifier (Femto, Germany) and digitised with a KPCI-3108 16-Bit A/D card (Keithley Instruments, USA) with a 1-kHz sampling rate. Single-pore recordings were processed in the programme QuB^{93 (link)}. The histogram of single-pore conductance (n = 100) was constructed by kernel density estimation (with the Gaussian kernel of 100-pS width) to overcome bin edge effects.